The studies proposed in this application are designed to determine the mechanism(s) involved in the in vitro inactivation of highly purified and cloned cytotoxic lymphocytes upon interaction with target cells in non-MHC restricted cytotoxicity reactions, as well as the metabolic events required for their reactivation. The specific aims are: 1) To Further characterize the recycling vs inactivation of highly enriched and cloned Tc and NK cells lines in in vitro cytotoxicity assays. 1a. To compare the recycling capacity of Tc cells in parallel MHC-restricted vs non- MHC-restricted cytotoxicity reactions to determine which effector cell and target ligands are required for recycling vs inactivation. 1b) To Investigate potential target cell specificity of NK cell inactivation. 2) To determine the biochemical mechanism(s) of effector cell inactivation. Highly enriched and cloned Tc and NK cells will be isolated after interaction with selected target cells. 2a) To Compare the target cell adhesion, recognition, triggering, and cytotoxic activity of cycled cells to control cytotoxic cells that have not been exposed to target cells. 2b) To Compare the levels of expression and/or chemical modification of specific proteins associated with cytolytic function for cycled cells to that of control cytotoxic cells that have not been exposed to target cells. 3) To investigate the cytokine requirements and metabolic events involved in the restoration of cytotoxic function to inactivated effector cells. 3a) To determine the kinetics of effector cell reactivation and the dependence on specific cytokines. 3b) To monitor the expression and activity of specific proteins associated with cytolytic function, as well as the ability of cells to respond signal transduction inducers during the course of reactivation. 3c) To analyze changes in gene expression that occur during the inactivation and reactivation of cytotoxic lymphocytes. The proposed studies of the mechanisms involved in the recycling vs inactivation of cytotoxic effector cells should provide valuable information about the regulation of cytotoxic activity that may have significant clinical applications.